Overview

Properties

Applications

Our Abpromise guarantee covers the use of ab3583 in the following tested applications.

The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.

Application Abreviews Notes
ICC/IF Use a concentration of 5 µg/ml.
Inhibition Assay Use at an assay dependent concentration.
ICC 1/50 - 1/200.
IHC-Fr 1/50 - 1/200.
WB 1/200 - 1/1000. Detects a band of approximately 16 kDa (predicted molecular weight: 16 kDa).
Flow Cyt Use 3-5µl for 106 cells.

Target

Images

  • ab3583 at a dilution of 1 / 4000 staining leptin in mouse 3T3-L1 cell lysate by Western blot.

  • ab3583 staining Leptin in 3T3-L1 cells by ICC/IF (Immunocytochemistry/immunofluorescence). Cells were fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton X-100 and blocked with 1% BSA for 1 hour at room temperature. Samples were incubated with primary antibody (2ug/ml in 0.1% BSA) for 3 hours at room temperature. An Alexa Fluor® 488-conjugated Goat anti-rabbit IgG (H+L) polyclonal was used as the secondary antibody (1/2000) (Panel a). Nuclei were stained with DAPI (Panel b). F-actin stained with Rhodamine Phalloidin (panel c). Merged images shown in panel d. 

  • ICC/IF image of ab3583 stained HeLa cells. The cells were 4% formaldehyde fixed (10 min) and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab3583, 5µg/ml) overnight at +4°C. The secondary antibody (green) was Alexa Fluor® 488 goat anti-rabbit IgG (H+L) used at a 1/1000 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43µM.

  • ab3583 staining Leptin in Human mesenchyml stem cells by ICC/IF (Immunocytochemistry/immunofluorescence). Cells were fixed with paraformaldehyde and blocked with 10% serum for 30 minutes at 22°C. Samples were incubated with primary antibody (1/100 in 10% horse serum) for 1 hour at 22°C. A FITC-conjugated Mouse anti-rabbit IgG1 monoclonal was used as the secondary antibody (1/100).

    See Abreview

  • Immunohistochemical staining of Leptin in Sheep brain using ab3583.

  • ab3583 staining Leptin in 3T3-L1 cells by Flow Cytometry. The sample was incubated with the primary antibody (3ug/ml in 2.5% BSA) for 2 hours at room temperature. An Alexa Fluor 488®-conjugated Goat anti-rabbit (1/400) was used as the secondary antibody. Red histogram represents ab3583, pink histogram represents isotype control, purple histogram represents unstained control, green histogram represents no primary antibody control

References

This product has been referenced in:
  • Han YC  et al. Leptin regulates disc cartilage endplate degeneration and ossification through activation of the MAPK-ERK signalling pathway in vivo and in vitro. J Cell Mol Med N/A:N/A (2018). WB . Read more (PubMed: 29372627) »
  • Liu Y  et al. HN1L Promotes Triple-Negative Breast Cancer Stem Cells through LEPR-STAT3 Pathway. Stem Cell Reports 10:212-227 (2018). Read more (PubMed: 29249663) »
See all 17 Publications for this product

Customer reviews and Q&As

1-10 of 18 Abreviews or Q&A

Abcam has not validated the combination of species/application used in this Abreview.
Application
Western blot
Sample
Spermophilus tridecemlineatus Tissue lysate - whole (white adipose tissue)
Gel Running Conditions
Reduced Non-Denaturing (Native) (12.5)
Loading amount
20 µg
Specification
white adipose tissue
Blocking step
BSA as blocking agent for 15 hour(s) and 0 minute(s) · Concentration: 3% · Temperature: 4°C

Abcam user community

Verified customer

Submitted Feb 05 2014

Answer

Thank you for contacting us. This antibody has not been tested for blocking functional activity. If you do choose to test it this we way encourage you to submit a review to let us and our customers know whether it worked for you in functional studies.

I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.

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Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Immunocytochemistry/ Immunofluorescence
Sample
Human Cell (Mesenchymal stem cell)
Specification
Mesenchymal stem cell
Fixative
Paraformaldehyde
Permeabilization
No
Blocking step
Serum as blocking agent for 30 minute(s) · Concentration: 10% · Temperature: 22°C

Mr. Steven Johnstone

Verified customer

Submitted Sep 05 2012

Answer

Thank you for your enquiry.

I can confirm that ab3583 and ab16227 antibodiesshould detect leptin both intra and extracellularlly.However based on the references for these product it appears that most if not all of the leptin is immediately secreted and would therefore be found extracellularlly.

I am sorry that as far as we are aware, these antibodies have not specifically been tested inchondrocyts.However, I would like to reassure you that our antibodies are tested and covered by our 6 month guarantee in the listed tested applications and species on the individual datasheets.

I hope this information is helpful. If you have any further questions, please do not hesitate to contact us.

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Answer

Thank you for passing some information to me.
I understand that ab3583 has been used previously at 1:4000.
Have you had a chance to test this antibody at dilutions of 1/500 or 1/1000 (for 1 hr at room temperature or overnight at 4oC?
Would you prefer to get a new vial of the primary antibody? Please do let me know how you wish to proceed.
I look forward to hearing from you soon.

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Answer

Thank you for getting back to me and for specifying the sample types.
As you have kindly explained to me in your previous e-mail, the primary and the secondary antibodies worked with the positive control but the signal was very weak using lysates from sheep cardiac tissues.
This antibody (ab3583) should recognize sheep Leptin, the cross-reactivity with this species has been tested and confirmed.
After reading through the detailed protocol you kindly forwarded to Abcam, I would like to make the following comments/suggestions:
1) Lysis buffer:
Leptin is secreted so potentially it can be degraded very quickly. It is very important to prepare the samples from fresh tissues and use lysis buffer which contains proteinase inhibitors. The whole process should be carried out on ice. I understand from the initial e-mail that Laemmli buffer was used for lysing the samples. I would rather suggest using RIPA buffer to prevent any unwanted protein degradation in the sheep cardiac tissues. The recipe of RIPA can be found at this website:
https://www.abcam.com/index.html?pageconfig=resource&rid=11379#A1
2) Loading amount:
Try to load at least 25-30 ug total tissue lysate per lane onto the gel.
3) Dilution:
You may need to optimize the dilutions of the primary antibody to find out the most suitable working dilution range for sheep cardiac tissue lysate. Brain tissue has high levels of leptin but cardiac tissue may have significantly lower. I have tried to find some information for you regarding the expressed levels of leptin in sheep tissue, but unfortunately not too much data are available on the public sites compared to human, rat our mouse (i.e. Swiss-Prot, Unigen).
http://www.ncbi.nlm.nih.gov/UniGene/clust.cgi?ORG=Oar&CID=524
http://www.ncbi.nlm.nih.gov/UniGene/ESTProfileViewer.cgi?uglist=Rn.44444
The expression levels of leptin may vary from cell type to cell type or from tissue to tissue. It would be worth testing 1/500 or 1/1000 (for 1 hr at room temperature or overnight at 4oC).
I hope this will be useful for you. Should you still have any problem with this antibody after following these suggestions, then please do not hesitate to contact me again. I would be delighted to help you further.
Have a nice day!

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Answer

Thank you for confirming the order dfetails.
Would you be so kind to clarify what samples you have been using:
- species,
- cell or tissue types,
- lysate (total cell lysates, or cytoplasmic fraction etc)
I look forward to hearing from you soon.

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Question


See below the answer to your question :
Q1: Could you please confirm the batch numbers of ab13987 and ab97051?
ab13987 : GR65976-1
ab97051 : GR44808-2
- Q2: Were these products shipped in the same package?
Yes I think so
- Q3: Could you please provide the Abcam Order Number(s) or your PON?
No we bought them from Sapphirebioscience in Australia, one of my
colleague ordered them, i have not these details.
- Q4: Have you optimized the dilutions of the primary and the
secondary antibody?
I did a dot blot to optimise the secondary dilution because the
recommended dilution of the primary (found in the datasheet) is
1:4000. For this dot blot instead of the 1:4000 dilution, I did a
mistake and use 1:800 dilution. The dot of protein was 300ng, 200 ng,
150ng, 100ng, 75 ng, 50 ng, 25 ng, 12.5ng. For the secondary the
dilution was 1:10000, 1:20000, 1:30000, 1:40000 and 1:50000. After a 2
min exposure, i was able to detect the 12,5 ng of protein with a
secondary dilution of 1:10000 and 1:20000; with the other secondary
dilution, i can detect 25 ng of protein (spot is very light) and 50
ng of protein with the same intensity as the spot obtained with 12,5
ng of protein with a secondary dilution of 1:10000 and 1:20000. Note
that the chemiluminescent detection system used for the dot blot was
more sensitive that the one used for the sensitivity test.
- Q5: Is the HRP still active (ab97051)? Have you used it successfully
with another primary antibody?
I think the antibody is still active because I used it beginning of
march for the dot blot and a week after to do a first western blot
with my protein lysate.
What i have done with this is antibody is:
1- the dot blot as describe above.
2- a western blot with my protein lysate (40ug total protein per lane)
with ab13987 as positive control (300ng), 1:800 dilution for the
primary antibody (I found out my mistake after the incubation),
1:20000 for the secondary (ab97051). I can't detect any band in my
protein lysate but i detected the positive control.
So we decided to do a sensitivity test to know what is the lower
amount of protein that the antibody is able to detect.
3- the sensitivity test
I hope to hear from you soon
Regards

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Answer

Thank you for your prompt answers. Your co-operation in this matter is highly appreciated.
I understand that you have already carried out some optimization steps and it seems that the primary and the secondary antibodies worked on positive control but the signal was very weak on the samples. Is that correct? Would you be so kind to clarify what samples you have been using:
- species,
- cell or tissue types,
- lysate (total cell lysates, or cytoplasmic fraction etc)
I have forwarded your response to our Australian Distributor (Sapphire) to get some confirmation about the shipment and order details. Could you please laise with them to find out the Abcam Order number?
Once again, thank you for your understanding and co-operation in this matter. I look forward to hearing from you and hope to solve this problem as soon as possible.

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Answer

Thank you for enquiry and for taking the time to provide some useful details of the experiments. As I understand three of our products (ab3583, ab13987 and ab97051) have been used in these studies.
Though you have kindly provided some details, it would be much appreciated if I could get some more information which would help me identify the source of the problem.
- Q1: Could you please confirm the batch numbers of ab13987 and ab97051?
- Q2: Were these products shipped in the same package?
- Q3: Could you please provide the Abcam Order Number(s) or your PON?
- Q4: Have you optimized the dilutions of the primary and the secondary antibody?
- Q5: Is the HRP still active (ab97051)? Have you used it successfully with another primary antibody?
Thank you for your understanding and co-operation in this matter. I look forward to hearing from you and hope to solve this problem as soon as possible.

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Answer

Thank you for contacting us.

I am sorry that the vial you received did not contain the full amount of the product and I apologize for the inconvenience.We will be pleased to issue a free of charge replacement vial. In order to do this, I am forwarding these details on to our distributors team who will be happy to organize this for you.

Thank you for your help and cooperation with this case. Please do not hesitate to contact us if you need anything further.

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1-10 of 18 Abreviews or Q&A

Please note: All products are "FOR RESEARCH USE ONLY AND ARE NOT INTENDED FOR DIAGNOSTIC OR THERAPEUTIC USE"

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